聚谷氨酸-顺铂复合物的制备及其生物活性

本文介绍了制备一种γ-聚谷氨酸-顺铂复合物，并考察其体外的抗肿瘤活性。主要通过生物发酵法获得γ-聚谷氨酸，酸降解法得到小分子γ-聚谷氨酸；利用PCR方法检测γ-聚谷氨酸-顺铂复合物对DNA的作用；利用MTT法来检测该复合物的体外抗肿瘤作用；利用流式细胞仪检测其对细胞凋亡的作用；利用小鼠体内实验检测其体内毒性作用。实验结果表明：成功获得γ-聚谷氨酸-顺铂复合物，该复合物载药率达10%～12%；该复合物对人肝癌细胞BEL7404、人非小细胞肺癌细胞H446和人结肠癌细胞RKO均具有显著的杀伤作用，能引起细胞凋亡(出现凋亡峰)；并且小鼠体内毒性试验表明该聚谷氨酸-顺铂复合物的毒性要比游离顺铂低。因此，γ-聚谷氨酸-顺铂复合物是一种有效的抗肿瘤药物，具有潜在的临床应用价值；生物发酵的γ-聚谷氨酸可用于药物载体，赋予药物新的特点。     

γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂含量测定方法的建立

目的建立γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂含量测定的方法。方法采用高效液相色谱法测定γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂含量,色谱柱为C18柱(4.6 mm×300 mm,5μm),流动相为含150mmol·L-1氯化钠的磷酸盐缓冲溶液(pH 7.4),流速为0.65 mL·min-1,检测波长为210 nm。结果顺铂质量浓度在4²00μg·mL-1范围内,其色谱峰面积与质量浓度的线性关系良好(r=0.999 7)。顺铂含量测定的加样回收率平均值为99.78%,RSD小于2%。测得γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂含量约为26%200μg·mL-1范围内,其色谱峰面积与质量浓度的线性关系良好(r=0.999 7)。顺铂含量测定的加样回收率平均值为99.78%,RSD小于2%。测得γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂含量约为26%²8%。结论该法灵敏度高、专属性强、可用于γ-聚谷氨酸-柠檬酸-顺铂复合物中顺铂的含量测定。     

新型棕榈酰谷氨酸-顺铂纳米粒的制备及其体外药效研究

目的以棕榈酰谷氨酸为载体材料制备新型高载药顺铂纳米粒(palmoyl glutamic acid-cisplatin-nanoparticles,PG-CDDP-NPs),并对其体外抗肿瘤活性进行研究。方法采用肖顿-鲍曼缩合法合成棕榈酰谷氨酸两亲性材料,以所合成的材料为载体,利用其与顺铂的配位作用制备高载药新型顺铂纳米粒,并对所制备的PG-CDDP-NPs性质进行考察;以市售顺铂注射剂为对照,采用MTT法考察了PG-CDDP-NPs对人肝癌细胞HEPG2的体外抗肿瘤活性。结果合成了棕榈酰谷氨酸,并成功制备了高载药量的PG-CDDP-NPs,PG-CDDP-NPs粒径为(106.33±4.45)nm,粒度分布较均匀,包封率为(84.20±0.81)%,载药量质量分数高达(27.03±0.12)%。体外释放研究表明,PG-CDDP-NPs具有缓慢释药性质,24 h累积释放量质量分数仅为(28.74±1.37)%。体外药效学实验结果显示PG-CDDP-NPs和CDDP-S对高表达的人肝癌细胞HEPG2细胞的IC50值分别为3.54μmol·L-1和4.55μmol·L-1。结论以棕榈酰谷氨酸为载体材料可制备高载药量的顺铂纳米粒;PG-CDDP-NPs在体外对HEPG2细胞有杀伤作用,与顺铂注射剂相比,显示出较强的抑瘤效果,PG-CDDP-NPs是一种具有潜在应用价值的顺铂纳米制剂。     

聚谷氨酸天冬氨酸顺铂复合物的体内毒性和抑瘤活性

目的:研究聚谷氨酸天冬氨酸顺铂复合物(PGA-Asp-CDDP)的体内毒性和抑瘤活性。方法:采用透析法、MMT法、昆明鼠和BALB/c裸鼠SMMC-7721肝癌模型检测PGA-Asp-CDDP的缓释作用,细胞毒性,体内毒性和抑瘤活性。结果:PGA-Asp-CDDP的10 h时顺铂累计释放率为32%,与人肝癌细胞SMMC-7721孵育72 h的IC50为(45.5±10.3),累计给药为12 mg·kg-1时体内毒性显著下降,并且对肝癌模型有显著抑瘤效果(P<0.01)。结论:聚谷氨酸天冬氨顺铂复合物有望成为治疗肝癌的一种安全有效的药物。     

葡萄糖脱氢酶缓释系统PL-γ-PGA纳米颗粒的制备及研究

γ-聚谷氨酸(γ-PGA)是一种由枯草芽孢杆菌发酵生产的阴离子聚合物,具有良好的吸水性、生物相容性和生物降解性,无毒性且可由人体降解代谢,是一种很理想的药物载体。发酵得到的γ-PGA一般都具有很高的分子量,本文通过酸水解的方法,得到分子量符合药物载体要求的γ-PGA,并采用离子凝胶法,在水相反应体系中由低分子量γ-PGA与聚赖氨酸(PL)制备得到形态及表面特性良好的PL-γ-PGA纳米颗粒,粒径200～500 nm。在此基础上,制备了搭载葡萄糖脱氢酶(PQQ-GDHs)的PL-γ-PGA纳米缓释系统,载药效率达到61.2%,并且具有很好的稳定性,在体外模拟胃肠道和血液系统的缓释结果表明,分别有31.7%和40.2%的PQQ-GDHs在前48 h被释放。     

产生聚γ-谷氨酸发酵的条件分析

微生物合成的聚γ-谷氨酸(γ-PGA)是一种可生物降解的,水溶性的聚合物,它将在食品,化妆品,涂料,土壤保护,医药等领域有很好的应用。本文利用本实验室筛选的枯草芽孢杆菌NX-2,探讨发酵制备聚γ-谷氨酸过程的最佳发酵条件。     

3-氨基苯甲酰胺联合顺铂抑制骨肉瘤细胞生长的作用

目的 探讨聚腺苷酸二磷酸核糖转移酶-1［poly(ADP-ribose)polymerase 1,PARP-1］抑制药3-氨基苯甲酰胺(3-aminobenzamide,3-AB)联合顺铂对骨肉瘤细胞U2OS的生长抑制作用. 方法 CCK-8法检测单独应用顺铂及与3-AB联合应用时U2OS细胞增殖的抑制情况,流式细胞术检测细胞凋亡情况并分析细胞周期. 结果 3-AB与顺铂联用的半数抑制浓度(IC50)比单独应用顺铂低,增敏比1.76,流式细胞术 结果显示3-AB联合顺铂处理后细胞的凋亡率较单纯3-AB、顺铂处理的凋亡率高,细胞周期分析 结果示3-AB联合顺铂处理较单纯顺铂处理S期阻滞时间更长. 结论 PARP 1抑制药3-AB能增强顺铂对骨肉瘤细胞的增殖抑制及促凋亡作用.     

顺铂明胶微球的研制及相关生物学特性

目的 研制包裹有顺铂的颗粒型可降解性栓塞剂。方法 采用改良的双相乳化冷凝聚合法制备顺铂明胶微球，以原子吸收光谱分析为基础，测定顺铂明胶微球的包裹率、载药率及体外释药特性。结果 顺铂明胶微球颗粒直径均匀，药物包裹率91％。载药率17.4%，体外4b内缓释96％。结论 本法制得的顺铂明胶微球性质稳定，微球降解速率及药物释放速度基本符合临床要求。     

固定化谷氨酸脱羧酶转化γ-氨基丁酸的研究

目的用固定化谷氨酸脱羧酶转化制备γ-氨基丁酸(GABA)。方法以海藻酸钠与明胶为协同作用载体包埋固定化酶,考察影响固定化酶活性的因素,获得固定化酶的优化条件,并将酶应用于制备GABA。结果海藻酸钠、明胶、氯化钙和戊二醛浓度分别为2.5%,1.0%,3.0%和0.3%,硬化时间4 h,固定化酶的相对活性最高,酶活回收率达到65.3%。以谷氨酸为底物,固定化谷氨酸脱羧酶为催化剂,采用填充床反应器连续制备GABA,连续反应60 h,酶相对活性仍保持在初始值的76.9%,谷氨酸转化率为98.7%。结论该固定化谷氨酸脱羧酶可应用于生物转化法连续制备GABA。     

顺铂脂质体的制备及其体内外评价

目的 制备抗肿瘤药物顺铂脂质复合物脂质体(CDDP-MM),以期降低顺铂的毒副作用,并保持其疗效。方法 使用逆相蒸发法制备CDDP-MM;以SD大鼠为实验动物考察CDDP-MM的体内药动学行为;以H₂₂荷瘤小鼠为模型,以市售注射用顺铂为参比,考察CDDP-MM的体内抗肿瘤效果及其毒副作用。结果 所制备的CDDP-MM平均粒径为109.8 nm,包封率高达90%。大鼠体内药动学结果表明,与注射用顺铂相比,CDDP-MM最大血药浓度(c_max)提高2.93倍(P<0.001),t_1/2α延长65.50倍(P<0.01);AUC_0-24显著增大41.26倍(P<0.01),CDDP-MM显著改善了注射用顺铂的体内药动学行为。小鼠体内抗肿瘤活性结果表明CDDP-MM的抑瘤率为82.5%,与注射用顺铂组相比小鼠体重显著增加(P<0.000 1),肾毒性下降。结论 CDDP-MM显著改善顺铂的体内药动学行为且可以有效抑制肿瘤生长、显著降低顺铂的胃肠道毒性和肾毒性。     

In vitro characteristics and in vivo plasma disposition of cisplatin conjugated with oxidized and dicarboxymethylated dextrans.

In vitro release behavior and cytotoxic activity, and in vivo plasma disposition of newly synthesized macromolecular derivatives of cisplatin (CDDP) were investigated and compared with CDDP. The derivatives included oxidized dextran conjugate of CDDP (OX-Dex/CDDP) and dicarboxymethylated dextran conjugate of CDDP (DCM-Dex/CDDP). In vitro release of platinum complex from dextran conjugated CDDP was determined by an equilibrium dialysis method. These dextran conjugates showed sustained release of the platinum complex. In vitro release half-life for DCM-Dex/CDDP was significantly longer (4.5 times) than that for OX-Dex/CDDP. In vitro cytotoxic activity of CDDP and dextran conjugated CDDP against colon 26, mouse colon cancer cell line, was measured using the MTT assay method. OX-Dex/CDDP showed a similar cytotoxic activity to CDDP. However, both cytotoxic activities were markedly decreased when preincubated with the medium containing serum. On the other hand, DCM-Dex/CDDP retained residual cytotoxic activity at a significantly higher level than OX-Dex/CDDP after preincubation with the medium containing serum, although it showed the lowest cytotoxic activity. This indicated longer maintenance of the in vitro antitumor activity of DCM-Dex/CDDP in serum compared with OX-Dex/CDDP. Plasma disposition of CDDP and dextran conjugated CDDP was determined by intravenous administration to rats. Although the total platinum plasma concentration-time profile for OX-Dex/CDDP was similar to that for CDDP, its markedly higher profile was achieved when DCM-Dex/CDDP was administered. The values of the total platinum AUC and MRT, where AUC is the area under the platinum concentration-time curve and MRT is the mean residence time, for DCM-Dex/CDDP were 11.2 times and 4.8 times significantly higher than with OX-Dex/CDDP in plasma, respectively. DCM-Dex/CDDP also showed a significantly lower total clearance compared with OX-Dex/CDDP. These results from the in vivo experiments revealed that retention of DCM-Dex/CDDP in blood circulation was much greater than that for OX-Dex/CDDP. DCM-Dex/CDDP thus has potential as a macromolecular derivative of CDDP for passive tumor targeting.     

PEGylated Multi-Walled Carbon Nanotubes for Encapsulation and Sustained Release of Oxaliplatin

Purpose

To develop PEGylated multi-walled carbon nanotubes as a sustained release drug delivery system.

Methods

Oxaliplatin was incorporated into inner cavity of PEGylated multi-walled carbon nanotubes (MWCNT-PEG) using nano-extraction. Oxaliplatin release rates from MWCNT-PEG-Oxaliplatin were investigated using dialysis tubing. Cytotoxicity of oxaliplatin, MWCNT-Oxaliplatin and MWCNT-PEG-Oxaliplatin were evaluated in HT29 cell by MTT assay, Pt-DNA adducts formation, γ-H2AX formation and cell apoptosis assay.

Results

Loading of oxaliplatin into MWCNT-PEG was ~43.6%. Sustained release occurred to MWCNT-PEG-Oxaliplatin, with only 34% of oxaliplatin released into medium within 6 h. In MTT assay, MWCNT-PEG-Oxaliplatin showed slightly decreased cytotoxic effect when cell viability was assessed at 12 and 24 h. A drastic increase of cytotoxicity was found when cell viability was assessed at 48 and 96 h. Pt-DNA adducts formation, γ-H2AX formation and cell apoptosis assay results showed the same trend as the MTT assay, suggesting sustained-release for MWCNT-Oxaliplatin and MWCNT-PEG-Oxaliplatin formulations.

Conclusions

PEGylated multi-walled carbon nanotubes can be used as sustained release drug delivery system, thus remarkably improving cytotoxicity of oxaliplatin on HT-29 cells.     

透明质酸修饰的空腔靶向纳米载体制备及其体内外性能评价

目的： 制备具有pH响应性的透明质酸衍生物修饰的主动靶向载药空腔纳米球载体,并进行相关性能测定。方法： 采用One-pot法制备空腔碳酸钙纳米球,并用透明质酸与壳聚糖的偶联物进行修饰,得到具有pH响应性的靶向给药载体;以阿霉素作为模型药物,对载体的粒径及Zeta电位、包封率、载药量及体外释放进行考察;以人肝癌HepG2细胞,通过MTT实验进行细胞毒性验证;以H22荷瘤小鼠为模型验证载体在体内及肿瘤部位的靶向性作用。结果： 所制备的靶向给药载体呈球状,平均粒径（376.8±12.4）nm,PDI为（0.295±0.080）,Zeta电位为（-45.1±0.3）mV,包封率（80.45±2.35）%及载药量（15.65±0.25）%;体外释放显示出此载体具有良好的pH响应性,且具有明显的缓释特征;细胞毒性实验证明了此载体具有低毒性;荷瘤小鼠实验证明了此载体具有良好的肝靶向和肝肿瘤靶向能力。结论： 成功制备了透明质酸衍生物修饰的碳酸钙空腔纳米球靶向给药载体,此载体具有良好肝癌靶向治疗的能力。     

Biodegradable Nanoparticles Containing Doxorubicin-PLGA Conjugate for Sustained Release

Purpose. Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] and the doxorubicin-PLGA conjugate was formulated into nanoparticles to sustain the release of doxorubicin. Methods. A hydroxyl terminal group of PLGA was activated by p-nitrophenyl chloroformate and reacted with a primary amine group of doxorubicin for conjugation. The conjugates were fabricated into ca. 300 nm size nanoparticles by a spontaneous emulsion-solvent diffusion method. The amount of released doxorubicin and its PLGA oligomer conjugates was quantitated as a function of time. The cytotoxicity of the released species was determined using a HepG2 cell line. Results. Loading efficiency and loading percentage of doxorubicin-PLGA conjugate within the nanoparticles were 96.6% and 3.45 (w/w) %, respectively while those for unconjugated doxorubicin were 6.7% and 0.26 (w/w) %, respectively. Both formulation parameters increased dramatically due to the hydrophobically modified doxorubicin by the conjugation of PLGA. The nanoparticles consisting of the conjugate exhibited sustained release over 25 days, whereas those containing unconjugated free doxorubicin showed rapid doxorubicin release in 5 days. A mixture of doxorubicin and its PLGA oligomer conjugates released from the nanoparticles had comparable IC₅₀ value in a HepG2 cell line compared to that of free doxorubicin. Sustained drug release was attributed to the chemical degradation of conjugated PLGA backbone, which permitted water solubilization and subsequent release of doxorubicin conjugated PLGA oligomers into the medium. Conclusions. The conjugation approach of doxorubicin to PLGA was potentially useful for nanoparticle formulations that require high drug loading and sustained release. The doxorubicin-PLGA oligomer conjugate released in the medium demonstrated a slightly lower cytotoxic activity than free doxorubicin in a HepG2 cell line.     

Fabrication of poly(γ-glutamic acid)-based biopolymer as the targeted drug delivery system with enhanced cytotoxicity to APN/CD13 over-expressed cells

Poly(γ-glutamic acid)-based targeted drug delivery system (PAMCN) targeting transmembrane metalloprotease aminopeptidase-N (APN/CD13) was fabricated and evaluated for the enhancement of targeting efficiency and cytotoxicity. The cisplatin (CDDP) loading content of PAMCN was about 36?±?5% and PAMCN showed a sustainable release profile with a half-maximal release time (t_1/2) of 23?h. The average size of PAMCN was 132?±?18?nm determined by light scattering (LS) and 158?±?67?nm by atomic force microscopy (AFM). Flow cytometry and fluorescence microscope analysis showed that the drug carrier (PAMN) could specifically bind to human umbilical vein endothelial cells (HUVEC). PAMCN enhanced the efficacy of CDDP to HUVEC cells with the half maximal inhibitory concentration (IC₅₀) value decreased to 90.83?±?33.00?μg/ml comparing with free CDDP treatment and showed less tube formation amounts (p?<?0.01) than free CDDP in matrigel angiogenesis inhibition assay in vitro. In vivo toxicity experiment indicated that the survival rate of KM mice in PAMCN group was 100% and PAMCN reduced the hepatic and renal toxicity significantly compared to free CDDP group. Therefore, this novel drug delivery system presents a promising potential for antiangiogenic chemotherapy.     

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: Microsphere-antibody conjugate

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71–141 μm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (～22–46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (～42–91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed ～54–70% and ～52–67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen–antibody activities were studied by immunofluorescence. Results indicated ～80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).     

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: microsphere-antibody conjugate

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71-141?microm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (approximately 22-46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (approximately 42-91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed approximately 54-70% and approximately 52-67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen-antibody activities were studied by immunofluorescence. Results indicated approximately 80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).     

温敏性吲哚美辛/β-环糊精包合物的制备及体外评价

目的合成兼具温敏性及药物包合能力的新型药物载体聚(N-异丙基丙烯酰胺)-β-环糊精(PNIPA-β-CD)，以吲哚美辛为模型药物，考察该载体的释药行为。方法末端带羧基的PNIPA与改性后的环糊精衍生物在1-(3-二甲氨基丙基)-3-乙基-碳二亚胺(EDC)的作用下缩合，得到PNIPA-β-CD，采用冻干法制备吲哚美辛/PNIPA-β-CD包合物。红外、¹H NMR和DSC表征载体的结构及包合物的形成；用分光光度法测定载体材料的LCST，并进行包合物体外释药研究。结果PNIPA-β-CD在35 ℃发生相转变，吲哚美辛/PNIPA-β-CD包合物的载药量为5.8％，药物与载体摩尔比为0.97∶1。体外释放研究表明吲哚美辛/PNIPA-β-CD包合物在37 ℃的释药比其在25 ℃的释放要慢，在LCST以上具有一定的缓释作用。结论该载体既具有温敏性，又具有药物包合作用，并且在体温条件下具有缓释作用，是一种新型的温敏性药物载体。